Project Summary We propose to study the effects of the chemical composition of the environment on the structure and function of intrinsically disordered proteins in the test tube and in the cell. The cellular environment is dynamic and heterogeneous in both space and time, and far removed from the idealized buffers commonly used in biochemistry experiments. How proteins function in this environment is a fundamental question in biology that is poorly understood. Especially puzzling are intrinsically disordered proteins, which make up over 30% of the human proteome and play a disproportionately large role in cellular regulation and misregulation. Intrinsically disordered proteins do not adopt a single native structure. Instead, they exist as an ensemble of conformations that have large surface areas and a low number of intramolecular bonds, making them highly susceptible to changes in solution composition. Why has evolution selected this malleable subset of proteins to act as central hubs and regulators of cellular function when routine cell cycle changes can alter their activity? Our lab aims to understand how disordered proteins function and interact in the complex cellular environment, and how the chemical composition of this environment can act as a master regulator that tunes their function. In order to do this, we use a multi-scale approach that combines all-atom simulations, high-throughput biophysical methods, and live cell microscopy. This bottom up study is designed to reveal the physical- chemical rules governing how solution-protein interactions affect protein structure and function. The broad goals of our work are (1) to gain a molecular-level understanding of how solution composition controls disordered protein structure and binding in the test tube and inside the cell; (2) to develop tools to control the chemical composition in the cell, and use this control to alter protein function; and (3) to identify and characterize the functional mechanisms of proteins that evolved to act as sensors and actuators of the cellular environment. The proposed work aims to uncover an entirely new ubiquitous regulation mechanism. Successful completion will uncover the underlying driving forces behind this solution-driven regulation, identify the proteins affected by it, and provide researchers with tools to leverage it towards control of cellular pathways. Most importantly, our findings will be based on physical chemistry principles, and so will not be organism-specific, and apply to a wide range of biomolecules, organisms, and ecosystems.